FI80600B - FOERFARANDE FOER FRAMSTAELLNING AV IMMUNOGENT PROTEIN- ELLER PEPTIDKOMPLEX. - Google Patents

Description

1 80600

A method for preparing an immunogenic protein or peptide complex

The present invention relates to a method of immunogenic complex comprising antigenic proteins or peptides having hydrophobic regions, having a complex spherical structure formed by circular subunits or portions of a spherical structure, and generally having a lower sedimentation constant than the corresponding micelles. and a higher sedimentation constant than the corresponding monomeric form of the protein or peptide, for preparation from amphipathic proteins or peptides or viruses, bacteria, mycoplasmas, parasites or animal cells containing them. The immunogenic complex between glucosides and proteins and peptides derived from viruses, mycoplasmas, bacteria, parasites or animal cells prepared according to the invention is the so-called "Iskomi". It can be used as a stimulant of the immune mechanism 20 as well as a vaccine.

It is known that killed viruses, for example influenza viruses, have a good antigenic effect. However, they are pyrogens even after a high level of purification. Isolation of components that are important in providing protective immunity has avoided the pyrogenic effect, but the immunogenicity is often not strong enough. Therefore, an appropriate adjuvant containing isolated antigens (subunits) had to be included in the vaccine to enhance the immune response. On the other hand, an effective adjuvant, when used as used so far, causes negative side effects. An adjuvanted vaccine is therefore no better than a vaccine based on the whole virus when it comes to pyrogenic effects.

2 80500

To increase immunogenicity, detergent-containing membrane proteins have been prepared that are placed on liposomes or attached to the surface of liposomes (EPC Application 794 008 30.0). Detergent-free membrane proteins in liposomes are described in U.S. Patent 4,148,876. The incorporation of an adjuvant into such detergent-free monolayer liposome products is described in U.S. Patent No. 4,196,197 (without explaining its effect). U.S. Patent 4,117,113 describes viral antigens containing 10 negatively charged liposomes. The incorporation of an adjuvant, mycobacterial cell walls, into liposomes containing influenza hemagglutination and neuramidase results in a better immune response. A better immune response is also obtained when an adjuvant such as killed Mycobacterium tuberculosis or Bortedella pertussis and saponins are introduced into negatively charged liposomes containing diphtheria oxide (U.S. Patent 4,053,585). However, all of the above lipid-containing membrane protein products are unstable upon prolonged storage, freeze-drying, or careless handling, for example, at high temperatures.

Protein micelles that are lipid-free and free of detergents have also been prepared as a vaccine. 25 Micelles of Semliki Forest virus (SFV) membrane proteins have been shown to stimulate the formation of circulating SFV antibodies and to protect the mouse against lethal SFV infection. On the other hand, such parainfluenza-3 virus membrane protein micelles were not effective in stimulating antibody production in sheep or in protecting against a dose of the PI-3 strain causing pneumonia unless the oil adjuvant was mixed with micelles, oil adjuvant, oil adjuvant which occur in the form of local reactions at the injection site (EPC application

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3 80600 811 022 13.6). The object of the present invention is to avoid these disadvantages and to provide a storage-stable protein or peptide complex with strong immunogenicity without side effects. This is achieved by forming a lipid-free complex between the hydrophobic regions of proteins and peptides derived from viruses, mycoplasmas, bacteria, animal cells or parasites, and hydrophobic groups located in one or more glycosides.

The method of the invention is characterized in that at least one of the above components is mixed with at least one solubilizing agent to form complexes between the amphipathic antigenic proteins or peptides and the solubilizing agent, after which the amphipathic antigenic proteins or peptides are separated from the solubilizing agent in the presence of a solubilizing agent. solubilized and transferred directly to a solution of at least one triterpene saponin with hydrophobic and hydrophilic regions at a concentration of at least the critical micelle content (CMC).

The new complex has a different morphological structure under an electron microscope than the corresponding protein model prepared without glycoside addition. Misel-25 is a dense central core with radially arranged branches, while the complexes prepared according to the invention have an open spherical structure consisting of circular subunits or parts of a spherical structure. The complex and its components still regularly have a lower sedimentation constant (see Figure 2) than the corresponding micelles and a higher sedimentation constant than the corresponding monomeric forms of the protein or peptide.

A complex prepared with a glycoside addition has better immunogenic activity than the corresponding protein micelles prepared without 4 80600 glycoside additions, or a complex between the protein molecule and a solubilizing agent (monomeric forms) or a lipid-bound protein molecule, e.g. viroso; and it provides fewer side effects than mixing 5 protein micelles with glycosides or a new ad-juvant. Thus, the dose of membrane proteins can be reduced to about 1/10 or more.

Proteins or peptides having hydrophobic regions that are complexed to hydrophobic regions located in glycosides can be: A) amphipathic proteins or peptides with hydrophilic and hydrophobic groups derived from or produced from enveloped viruses, mycoplasmas, bacteria, parasites, parasites - 15 teens or peptides or such proteins or peptides; B) hydrophilic proteins or peptides which are rendered amphipathic by attaching hydrophobic groups to them. These proteins or peptides may be derived from viruses, mycoplasmas, bacteria or parasites; C) amphipathic proteins or peptides obtained by making the inaccessible hydrophobic portions of the hydrophilic proteins chemically accessible. These proteins may be derived from the aforementioned microorganisms or cells.

a) In the case of membrane proteins or peptides derived from whole cells or viruses, the preparation of the complex basically comprises the following steps: purification or isolation of animal cells or microorganisms or fragments thereof; and dissolving the hydrophobic proteins and removing the solvent when the desired antigen is introduced at the same time

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5 80600 complex via a glycoside in immunogenic form (immunogenic complex).

The enveloped virus, mycoplasma, bacteria, parasites, and animal cells are concentrated and purified in 5 known ways (see The Tools of Biochemistry, TG Cooper, John Wiley & Sons (1977) New York; incorporated herein by reference), e.g., by filtration, ultrafiltration, or electrophoresis and various by chromatographic methods, for example by gel filtration or gradient centrifugation through a sugar-10 gradient or by hollow fiber dialysis. In the case of bacteria, it may be necessary or advantageous to first dissolve or break the cell walls (see Cota-Robles and Stein; CRC Handbook of Microbiology, Part II (1973), pp. 833-844; incorporated herein by reference) e.g. using ultrasonic or French press treatment.

The purified animal cells, microorganisms or fragments thereof are mixed with this nonionic, ionic or zwitter ion detergent or bile acid derivative detergent 20 which is used in excess. Typical examples of suitable nonionic detergents are polyglycol esters and ethers formed with aliphatic and aralylate acids and alcohols. Examples are alkyl, polyoxyethylene ethers of the general formula CnH2 +1 (OCH2CH) XOH, abbreviated

CnΕχ; alkyl polyoxyethylene ethers with a phenyl ring between the alkyl group and the polyoxyethylene chain, abbreviated Cn 0 Εχ, e.g. -Ex, e.g. Tween 20 or Tween 80, β-D-alkyl glycosides, e.g. β-D-octyl glycoside. In addition to the above-mentioned glycosides, saponin in particular can be used. However, these are weak detergents and should be used in combination with other detergents. Typical examples of suitable ionic detergents are bile acid detergents such as deoxycholate and cholate. Conjugated detergents such as taurodeoxy-5 cholate, glycodeoxycholate and glycocholate may also be used. Lysolecithin as well as synthetic lysophosphides can be mentioned as Zwit-ter ionic detergents. Mixtures of the above detergents can also be used.

Dissolution can be performed with alcohols, organic solvents, or small amphipathic molecules such as heptane-1,2,3-triol, hexane-1,2,3-triol, acetic acid, water-soluble peptides and proteins, or mixtures or detergents thereof.

The solvent is used in excess relative to the amount of lipid and hydrophobic proteins. The cells or microorganisms and the solubilizing agent are suitably mixed in a weight ratio of 1: 3 to 1:10.

The cells or microorganisms and the solvent are mixed in a buffered, possibly saline, solution. The molarity of the saline solution is between 0.02 and 0.5 M, preferably between 0.05 and 0.25 M, preferably between 0.1 and 0.2 M. The detergent should act for about 1 hour at room temperature.

The common salt is considered to be the preferred salt, but other salts may also be considered, especially those formed by alkali and alkaline earth and ammonium ions, as well as strong mineral acids and organic acids such as acetic acid, trichloroacetic acid, formic acid and oxalic acid. Suitable buffers are all substances which are buffered in the pH range from 6.5 to 9. When using cholates and deoxycholates, a pH range from 8 to 9 is preferred, and when using nonionic detergents, a pH of 7.4 is preferred. When using organic acids to dissolve proteins, the buffer can be omitted.

7 80600

When dissolving cells or microorganisms, a mixture of solvent and cell or microorganism fragments (fragments mentioned below) is formed. Among the fragments are charged, monomeric antigen proteins with hydrophobic regions in a complex formed with solvents. The novel immunogen complex is prepared by separating or directly attaching the charged monomeric antigen proteins from the solvent in the presence of one or more glycosides, wherein the glycosides 10 must have hydrophobic and hydrophilic regions and be present at a concentration that is at least critical for micelles. Other fragments are removed before the complex is prepared according to the invention, while at the same time as or after, but before.

The complex can be prepared either by removing the solvent from, for example, dialysis, gel filtration, or chromatography from a mixture of solvent, charged, monomeric antigen proteins, glycoside, and optionally other fragments, or by separating charged, monomeric antigen proteins from said mixture, e.g. , by chromatography or electrophoresis. According to the invention, it is essential that the monomeric antigen proteins are separated from the solubilizing agent in the simultaneous presence of a glycoside, or that, after separation, they are attached directly to the glycoside, which must be in micelle form.

When the monomeric antigen proteins are separated from the solubilizing agent so that they can come into direct contact with the glycoside, a special complex is formed. The micellar form of the glycoside is thought to form a complex, and it is assumed that this is formed by the attraction between the hydrophobic regions of the glycosidic cells and the hydrophobic regions of the membrane proteins. The amount of glycoside 8 in the complex varies depending on the glycoside used and the complexed membrane proteins, and is about 0.5 to 50% by weight, especially 0.5 to 25% by weight, mainly 0.5 to 15% by weight, often 0.5 to 10% by weight, in particular 2 to 8% by weight. In contrast, if the charged antigenic monomeric proteins are separated from the solubilizing agent in the absence of a glycoside, protein species of the type prepared according to EPC 811 022 13.6 are formed.

It is convenient to remove other fragments by gradient centrifugation because the sedimentation constants of said components decrease in the following order: cell fragment, protein complex with solubilizing agent or glycoside, monomeric proteins, and solubilizing agent. Thus, other fragments can be removed by gradient centrifugation from a mixture of solvent, monomeric proteins, and other fragments before the glycoside is added and the solvent removed, e.g., by dialysis, gel filtration, or chromatography, or the monomeric proteins are separated from the solvent, e.g.

by electrophoresis, chromatography or gradient centrifugation. In the latter method, it is also possible to remove other fragments by the same gradient centrifugation during which the complex is formed. It is also possible to separate the other cellular components after the complex is formed as described above, e.g. by centrifugation, affinity chromatography or gel filtration.

The glycoside is triterpene saponin. Saponins are described in R. Tschesche and Wulf's Chemie und Biologie der Saponine; Fortschritte der Chemie organischer Naturstoffe, published by W. Herz, H. Grisebach and G. W. Kirby, vol. 30 (1973). Preferably, highly polar saponins are used, mainly polar triterpene saponins, e.g. a saponin extract obtained from the aralocid A of the bark of the discipline 980600, Chikusetsu-saponin IV, Ca-lendula-glycoside C, Chikusetsu-saponin V: Achyranthes saponin B, Calendula glycoside A, Aralocid B, Aralocid C, Putranjia saponin 5 HI, Bersama saponoside, Putranjia saponin IV, Trichosid A, Trichosid B, Saponasid A, Trichosid C, Gyposid, Nutanoside, Dianthosid C, Saponasid D, mainly aeskin obtained from Aesculus hippocastanum [T. Patt and W. Winkler: Der 10 therapeutisch wirksamme Prinzip der Rosskastanie (Aesculus hippocastanum), Arzneimittelforsch. 10 (4), (1969), p. 273-275], or sapoalbine obtained from Gypsophilla struthium [R, Vochten, R. Joos and R. Ruyssen: Physicochemical properties of sapoalbin and their relation to the foam stability, J. Pharm. Belg. 42, (1968), ss. 213-226], especially the saponin extract obtained from Quillaja saponaria Molina, primarily a DQ extract prepared according to K. Dalsgaard [Saponin Adjuvants, Bull. Off. Int. Epis 77 (7-9), (1972), p. 1289-1295], and 20 Quil A prepared according to K. Dalsgaard [Sapon Adjuvants III, Archie filr die Gesamte Virusforschung 44, (1974), p. 243 - 254]. Mixtures of glycosides may also be used. The amount of glycosides used should be at least 1-3 times their critical micelle-forming concentration (VMC), preferably at least 5, especially at least 7-12 times this amount. It is assumed that the glycoside can then be bound to and encapsulated in the monomeric forms of the membrane proteins. Preferably, Quil A is used with a critical micelle-forming concentration of 0.03% by weight. The amount of Quil A should be at least 0.02% by weight, in particular 0.05 to 0.5% by weight, in particular 0.2% by weight. The publications on the above glycosides are incorporated by reference.

ίο 80 600

Separation of charged, monomeric antigen proteins from the solute has been accomplished by centrifugation, dialysis, electrophoresis, and various chromatographic methods.

Mixtures prepared as described above containing microorganisms corresponding to fragmented cells and a solubilizing agent are centrifuged and layered, for example, on a sugar or saline solution containing a solubilizing agent, followed by a glycoside-containing gradient such as a sugar gradient or a glycerol gradient or a meridian gradient. or a heavy salt, e.g. CsCl (i.e. relatively inert substances having a suitable density and viscosity to act as a gradient material) e.g. e.g. in the concentrations given for the sugar gradient below.

The gradient system is centrifuged to at least 100,000 g. The sugar may be monosaccharides or disaccharides, such as lactose, maltose or sucrose, but trioses, tetroses and glycerin are also used. 20 Sucrose is mainly used. A suitable initial concentration of the gradient sugar concentration is at least 5%, predominantly 15-25% by weight (top in the gradient) and a final concentration of at least 20%, predominantly 45-60% by weight (lowest in the gradient). For example, the gradient may consist of 25 upper layers with a sugar content of 5 to 25% by weight and a lower layer with a sugar content of 20 to 60% by weight. However, the gradient may also have several layers, whereby the concentration differences of the separate layers decrease to a corresponding degree. The sugarigra-30 orientation contains a glycoside or a mixture of glycosides. The amount of glycoside should be at least 1-3, preferably at least 7-12 times CMC, Quil A at least 0.02, especially at least 0.05-0.5, preferably at least 0.2% by weight. In this glycoside-containing gradient, a solute of 80,600 is separated, and the complex between this and the protein is converted to a protein-glycoside complex.

Above the sugar gradient is a layer of solution consisting of sugar or heavy salt and containing a solvent or a mixture thereof. Lipids remain in this layer. The concentration of the solvent in this layer is less than or equal to the superimposed mixture of microorganisms or cells and the solvent, and is suitably between 0.12 and 3% by weight, preferably 0.75% to 1.5% by weight, whereby 1% by weight is preferred. The concentration of salt and salt may be the same or it may be lower than the concentration of the upper layer of the lower glycoside gradient.

After centrifugation of at least 15 100 000 g for at least 16 hours, in particular for 20 hours at 20 ° C, the proteinaceous fractions are collected and dialyzed against buffer; (0.5 to 0.001 M), primarily against 0.005 M Tris-HCl, 0.01 M NaCl (pH 7.4) or 0.2 M anunonium acetate buffer (pH 7.0) and concentrated as an example. as described by TG Cooper in The Tools of Biochemistry (ed. John Wiley & Sons, New York, 1974; incorporated herein by reference), for example, by lyophilization, vacuum dialysis, or ultrafiltration. During centrifugation, all components are sedimented, releasing the solute from the complex it forms with the protein and transferring the monomeric proteins to the glycoside with which they form a complex. Subsequent dialysis removes the sugar. Optionally, the complex can be further purified, e.g., by gradient centrifugation with respect to the free Gly-30 coside, e.g. with a sugar gradient containing 25 to 60% by weight of sugar, preferably 10 to 40% by weight of sucrose.

i2 80 600

Alternatively, after purification of the microorganisms or cells as described above and after mixing the cells with the solvent in the above weight ratio, 1-3, preferably at least 7-12, of the cells and the solvent in the above buffer may be added directly. times the amount of CMC, for Quil A 0.05 to 2% by weight of glycoside, predominantly 0.2% by weight of glycoside, and dialyze against a buffer of 0.5 to 0.001 M, predominantly 0.005 M Tris-HCl, 0.01 M NaCl (pH 7.4) or 0.2 M upstream

ammonium acetate buffer (pH 7.0). The solvent is then removed in the presence of the glycoside. The prepared membrane-protein complex is then isolated by sedimentation gradient centrifugation as described in the last paragraph of page 10, but without the addition of glycosides, and other fragments as well as free glycoside are removed.

The mixture of cells or microorganisms and the solubilizing agent in the buffer can also be centrifuged at 20 and e.g. layered on a 5-60% by weight sugar gradient prepared in the above-mentioned buffer, mainly a 10-20% by weight sucrose gradient, and centrifuged at least 150% by weight. 000 g for at least 20 minutes, mainly for 30 minutes at 250 000 g. In this case, the remaining fragments differ from the complex formed by the solubilizing agent and the protein.

The surface-containing proteinaceous liquid, designated the peak fraction, is removed and glycoside is added in an amount of at least 1-3, preferably at least 7-12 times CMC, 0.05 to 0.5% by weight for Quil A, preferably 0.2% by weight, and dialyzed against 0.5 to 0.001 M buffer, especially 0.005 Tris-HCl, 0.01 M NaCl (pH 7.4) or mainly 0.2 M ammonium acetate. In this case, the solubilizing agent is removed in the presence of the glycoside. Additional 35 purification can be performed by sedimentation gradient centrifugation (see page 10, paragraph 2). Further purification can take place by centrifugation through a sugar gradient containing 5-60% by weight of sugar, mainly 19-40% by weight of sugar.

Alternatively, a mixture of fragmented microorganisms or cells and a solubilizing agent or proteinaceous centrifuge (other fragments and free solubilizing agent are absent) obtained by gradient centrifugation of this mixture in a buffer of 10 to 60% by weight can be obtained. , preferably through a sugar gradient of 10 to 20% by weight, is separated electrophoretically from the solubilizing agent and introduced into a solution containing at least 1 to 3, preferably at least 7 to 12 times CMC, 0.05 to 0.5% by weight for Quil A -% glycoside, preferably 0.2% to 15% by weight of glycoside. In this case, the charged, monomeric antigen proteins are separated from the solubilizing agent. In the case of separation by electrophoresis, it is suitable that the solvent-buffer solution does not contain additional salt addition, which can interfere with the electrophoresis and produce too high a temperature.

For example, zone electrophoresis with or without a carrier and iso-tachophoresis with or without a carrier can be used. As carriers, conventional substances such as polyacrylamide, agar, silica gel, starch, cellulose, polyvinyl chloride, ion exchanger or celite can be used. Isolation and concentration of the complex is performed as described on page 11, lines 20-23. See page 11, lines 28-32 for additional purification by gradient centrifugation.

If the starting material contains hydrophobic membrane proteins with different charges or weights, these can be separated by electrophoresis or centrifugation methods and their separate complexes prepared.

i4 80600 This method can thus be used to separate and enrich different membrane proteins.

Optionally, after purification from the cell fragments, the dissolved proteins can be separated from the solubilizing agent by chromatographic methods, e.g., gel filtration, with the antigenic structures adsorbed on an insoluble support (matrix), which can be, for example, cellulose, agarose, dextran, acrylamide, and glass. Different ligands are coupled to the matrix structure, which then acquires different properties that are utilized in the isolation. Usually, the antigenic structure is adsorbed at the same time as the solvent used passes through the matrix without adsorbing it. This is followed by the release of antigen 15. Before or during the release step, there may be an exchange of solvent, salt and buffering agent, in which case the solvent may be replaced by a glycoside and the complex prepared.

In ion exchange chromatography, charged ligand molecules such as diethylaminoethyl (DEAE) are coupled to a matrix and used as a cation exchanger. Carboxymethyl (CM) or phosphate groups (P) are attached to the matrix and used as an anion exchanger. Utilizing the difference in antigenic structures and net solvent charges, separation of these molecules is achieved. Usually the solubilizer is uncharged and the protein is charged. Elution is carried out by means of a salt gradient, e.g. K or NaCl, or by adjusting the pH with a suitable buffer, e.g. phosphate buffer, in the presence of a solubilizing agent (see concentration and example in connection with the above dissolution). During elution, the proteins can be purified, the solvent changed, or complexed if a glycoside is added to the eluent instead of the solvent. The salts are removed after -35 by dialysis or gel filtration.

is 80 600

Gel filtration takes advantage of the fact that the solubilizer is smaller in size than the antigenic structures and that it comes out in subsequent fractions, as the complex forms, the size of the antigen-containing structures 5 increases, and the zones containing the solubilizing agent increase.

The antibodies can be irreversibly bound to the above matrix by immunoaffinity chromatography, after which the unique specificity and affinity of the antibodies are exploited to purify the desired antigenic structure. The solvent has no affinity for the antibodies. Elution takes place by slight denaturation, for example by lowering the pH to about 4, and in the presence of a solvent or glycoside.

Lectin chromatography utilizes lectins, a group of proteins that can react reversibly with specific sugar groups, allowing them to bind to, for example, glycoproteins. The lectin is coupled as a ligand, for example

Sepharose (Pharmacia, Uppsala) or purchased as a pre-assembled, commercial product in a suitable matrix. Detergents (solvents) have no affinity for coupled lectin. The adsorbed antigenic structures are usually cleaved by the addition of methylated sugars, for example, methyl mannoxide, methyl glucoside, and N-acetylglucosamide dissolved in buffered saline in the presence of a solubilizing agent or glycoside.

In covalent chromatography, the antigen-30 structure is covalently attached to the matrix by a thiol group. The thiol group in the activigene is selectively linked to an activated thio group attached to a suitable matrix by thiodiosulfide exchange. This bond is reversible, and after the solvent 35 is washed away, the thiol-binding antigen structures can be eluted by reducing the disulfide bond with mercaptoethanol or dithiothreitol in the presence of a solvent or glycoside.

Hydrophobic Chromatography: This technique utilizes the interaction between an octyl- or phenyl-type, immobile, hydrophobic ligand and the hydrophobic surfaces of antigenic components. Alternatively, this technique may be a method of binding the solubilizing agent from the mixture to the ligand at the same time as the non-adsorbed antigenic structures can be recovered for further work according to Example 4 (dialysis method). In other conditions, the antigenic structure can be bound to the ligand, and the solubilizing agent has no affinity for the ligand, so the min-15 is continued according to the dialysis method. The coupling is performed with a high ionic strength, e.g. ammonium sulphate, and eluted with a low ionic strength, e.g. water or ethylene glycol.

When the complex contains bacterial membrane protein-20 mines, it is preferable to first break the cell walls before treating the cellular material according to the method described above. Examples of bacteria from which hydrophobic proteins can be prepared are, for example, Escherichia, Staphylococci, Haemaphilus, e.g. H. influenzae, Bordetella, e.g. B. pertussis, Vibrio, e.g. V. cholerae, Salmonella, e.g. S. typhi and S. paratyphi, mainly Colin, e.g. pili K 88, adhesion factor and Salmonella pore protein or outer membrane proteins of B. pertussis and Neisseria meningitidis.

Examples of useful enveloped viruses are Orthomyxoviridae such as influenza A, B and C, Paramyxoviridae, primarily measles virus, mumps virus, parainfluenza 1, 2, 3 and 4 virus, rabies virus and rinderpest virus, in particular vesiculovirus, Rhabdoviridae, feline leukemia- 11 i7 80 600 virus and bovine leukemia virus, Herpesviridae, primarily Pseudorabies, Coronaviridae, Togaviridae such as EEE, WEE and VEE (Eastern, Western and Venezuela equine encephalitis), yellow fever virus, especially bovine viral diarrhea virus 5 and European swine fever virus -dae, Bunyaviridae, Iridioviridae, especially African swine fever virus, and among the unclassified viruses are human hepatitis B virus and Marburg / Ebola virus.

Examples of parasites that can be used according to the invention are Protozoa, such as Toxoplasma, e.g. Toxoplasma gondii, Plasmodium, e.g. Plasmodium vi-vax, malariae and falciparium, Teileria parvum and ovale, and Filaroidae, mainly Parafilaria and Ochoocerca, En-tamo , various anaplasmas, Schistosoma, such as Schistosoma haematobium, mansoni and japonicum, and Trypanosoma, e.g. Trypanosoma gambiensi, T. brumseo or congolesi.

b) It is also possible to start from hydrophobic non-membrane proteins or non-hydrophobic proteins or peptides. Non-hydrophobic proteins or peptides can be converted to hydrophobic by attaching hydrophobic groups to them. Said proteins may be derived from enveloped or non-enveloped viruses, bacteria, mycoplasmas or parasites. Examples of viruses that do not have an envelope or hydrophobic proteins are Picornaviridae (which is also considered to have hydrophobic proteins), e.g. foot-and-mouth disease virus, poliovirus, Adenoviridae, Parvoviridae, e.g. feline plague virus and porcine parvovirus, Reotaidae, e.g. -virus. Examples of mycoplasmas are M. Pneumoniae, Mycoides, bovis, suis, hyorinos, orale, salivarium, hominis and ter-mentans.

These proteins and peptides are purified as described in a); cleaning and insulation.

35 ie 80 600

Hydrophobic groups that can be attached to non-hydrophobic proteins are straight, branched, saturated or unsaturated aliphatic chains having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, preferably 6, 7 or 8 carbon atoms; small peptides of 1, 2, 3, 4 or 5 amino acids, preferably 2, 3 or 4 amino acids, which are Trp, Ile, Phe, Pro, Tyr, Leu and Var, preferably Tyr; a cholesterol derivative such as cholinic acid or ursodeoxycholinic acid.

These hydrophobic groups should be attached to a group that can be attached to a non-hydrophobic protein, such as carboxyl, amino, disulfide, hydroxyl, sulfuryl, and carbonyl groups, such as aldehyde groups.

Hydrophobic linking groups are mainly carboxyl, aldehyde, amino, hydroxyl and disulfide derivatives of methane, ethane, propane, butane, pentane, hexane, heptane or octane, as well as peptides containing Cys, Aspin, Glu. Lys is primarily octanal and Tyr-Tyr-Tyr-Cys, -Aspin or -Gluin. Hydrophobic groups having a linkable group must be dissolved in water, e.g. with the above-mentioned solubilizing agents and detergents, or with hydrochloric acid, acetic acid, glacial acetic acid, lye or ammonia, depending on the substance to be dissolved. The pH is then adjusted to the neutral range without precipitation of the substance, taking into account that no pH is obtained which denatures the protein to which the hydrophobic group is to be attached.

Hydrophobic molecules are attached to the non-hydrophobic protein in a molar ratio of 10 to 0.1: 1, primarily 1: 1.

Hydrophobic groups in which the linking group is a carboxyl group can be attached to proteins via water-soluble carbodiimides or assembled anhydrides. In the first case, the carboxyl group is activated at pH 5 with carbodiimide and mixed with protein 35 dissolved in a buffer having a pH of 8 and a high phosphate content. In the latter case, the carboxy compound is placed with isobutyl chloroformate in the presence of triethylamine in dioxane or acetone triil and the resulting anhydride is added to the protein at pH 8 to 9. It is also possible gives hydrazone bonds.

10 Amino groups can be converted to diazonium salts at low temperature by means of nitric acid, which give azo bonds with Tyr, His and Lys.

Hydroxyl groups can be converted by means of succinic anhydrides to hemisuccinate derivatives which can be coupled as carboxyl groups.

Aldehyde groups can be converted to Sohiff's base by amino groups in the protein.

Many coupling groups and methods are described in Journal of Immunological Methods, 59 (1983), 20, pages 129-143 and 289-299; Methods in Enzymology, Vol. 93, pp. 280-333, and Analytical Biochemistry 116, (1981), pp. 402-407; which are incorporated herein by reference.

The proteins and peptides thus prepared, which have obtained hydrophobic groups, are complexed with the glycoside described in a), but the purification step in which cell fragments are removed can be omitted.

c) It is also possible to start with hydrophilic proteins encapsulated with hydrophobic groups and to access the latter by denaturing the proteins mildly, for example at low pH, at about pH 2.5, with 3-M urea or at high pH. at a temperature above 70 ° C. Such proteins may be immunoglobulins such as IgG, IgM, IgA, IgD and IgE. Im- 80 80,600 immunoglobulins can be used as anti-idiotypic antibodies. The proteins are further purified as the proteins in b) and then complexed to the glycoside as described in a), omitting the purification step 5 to remove cell fragments.

Immunogen complexes can be used to specifically stimulate the immune system in humans and animals. They can thus be used as a vaccine against diseases caused by bacteria, mycoplasma, viruses and parasites, and when the aim of the study is to produce antibodies against membrane proteins of various animal cells.

Mixtures of amphipathic proteins or peptides from different bacteria or viruses can also be used when it is desired to prepare a vaccine against several diseases.

The complexes prepared according to the invention can be used for human and veterinary compositions containing isochoma, optionally together with customary additives and diluents, preferably isochoma in the form of a buffer solution, e.g. in its TN solution (see Example 1).

The invention is described in more detail by the following working examples.

25 Test 1

Comparison between the membrane protein complex prepared with Quil A according to the invention and the immunogenic activity of the corresponding membrane protein cells prepared without glycoside addition.

30 mice (BALB C, Bonhollgard, Denmark; weight about 20 g) were divided into 5 groups and the groups were immunized twice every three weeks with buffer alone (PBS), 0.1 pg or 5 pg of the complex prepared using influenza-derived membrane proteins 35 as well as Quil A, 100 in silicon PBS according to Example 1 and 2i 80,600 using 5 pg of glycoside-free membrane protein micelles prepared according to EPC application 811 022 13.6.

Serum was collected from mice each week until the end of the experiment tree. Serum immune response was measured by ELISA (Voller, Bidwell, & Bartlett 1980, Enzymelinkes Immunosorbent Assay, pages 359-371; Manual of Clinical Immunology, American Society for Microbiology, Washington, USA).

The result is shown in Figure 6. It shows that the antigen complex prepared with Quil A gives a better immune response than the corresponding protein micelles without the addition of Quil A. A protein-name membrane complex prepared with 0.1 pg of Quil A elicits a higher immune response than 15 pg of glycoside-free protein micelles.

Test 2

Comparison of the immunogenic activity and side effects of the membrane protein complex prepared according to the invention with Quil A and the corresponding membrane protein micelles mixed with a glycoside as adjuvant additive.

The experiment uses 30 guinea pigs bred by the SVA (State Veterinary Institute). They are divided into 5 groups of 6 animals each and vaccinated with influenza virus (strain Soiva) protein protein (P) or a membrane protein complex from the same influenza virus prepared with glycoside Quil A (GP) according to Table 1. . Preparation according to Example 2. 6 guinea pigs were unvaccinated controls. The GP complex is released from the free glycoside by sucrose gradient centrifugation. The results are shown in Table 1. P-micelles as well as the GP complex give minimal reactions and the PG-35 complex gives clearly higher antibody responses 22 80 600 than the P-micelles. A mixture of 10 pg of free glycoside as well as β-micelles elicits mild reactions but no adjuvant effect. The addition of 100 pg of free glycoside gives strong local reactions in the form of erythema and edema, as well as a higher antibody response than the P-micelle vaccine without the free Quil Aita.

table 1

Immune response to influenza virus in guinea pig see-10 rum as measured by the haemagglutination inhibition (HI) test after two vaccinations every 4 weeks. The serum is taken 10 days after the second vaccination.

P = 1 pg protein micelle consisting of influenza virus-15 peplomers (H and N) P + 10 = 1 pg protein micelles + 10 pg free glycoside P + 100 = 1 pg protein micelles + 100 pg free glycoside 20 GP = 1 pg protein complex made with glycoside containing <0.1 pg glycoside - no reaction + slight flushing ++ moderate flushing 25 +++ severe flushing and necrosis HI titer (2log / l)

P P + 10 P + 100 GP

M ± SD 6 animals 7.9 ± 2.3 7.5 ± 1.5 10.3 ± 3.1 9.7 ± 0.6

Local reaction - + +++ 30 [(In unvaccinated guinea pigs the titers were 2 (6 animals)]. 35 23 80600

Test 3

Membrane protein complexes prepared according to Example 1 and further purified by centrifugation through a 10-40% sucrose gradient were tested for immunogenic activity. There are no detectable amounts of free Quil A (<0.02% by weight) in this preparation as measured by the cell lysing activity of Quil A. Cell lysing activity was examined in bovine red blood-10 bones. Blood cells were run on an agarose gel at a concentration of 0.05%. The agarose gel was placed in an electrophoretic system in which Quil A moves toward the anode and dissolves blood cells. The dissolved zone has a rocket shape and the amount of glycoside is proportional to the length of the rocket, and is visualized by protein staining with Coomasie Brilliant. This method can determine a glycoside concentration of 0.02% or greater [Sundqvist et al., Biochemical and Biophysical Research Communications, Vol. 114 (1983), pages 699-704]. 20 In sedimentation analysis, the further purified material has the same sedimentation constant as the unpurified material. Electron microscopy detects particles with the same morphology (see Figures 2 and 3). In the case of mouse vaccination, the material was as active as the crude material in the case of antibody stimulation. In one experiment, a 1 pg or 0.1 pg influenza virus virus protein complex prepared with Quil A addition and purified by two gradient centrifugations as described above was as effective as the corresponding, still unpurified material. The above result indicates that the complexes are stable and potent immunogens without detectable amounts of free Quil A.

24 80600

Experiment 4 3 different vaccines with Hepatitis Bs (HBs) antigen were each tested in 4 mice using a 5 pg dose of HBs antigen. These 3 vaccines were: is-5 comas prepared according to Example 14; Intact particles of 22 nm corresponding to a commercially available vaccine and an experimental vaccine in which the corresponding HBs proteins are in the form of a micelle prepared according to EPC application 811 022 13.6. The antibody response has been assessed by ELISA.

Table 2

Mouse serum antibody response to HBs antigen as measured by ELISA 15

Iskomit_22 nm particles_cellules 1.343 0.603 0.569 1.788 0.598 0.240 1.841 0.888 0.273 20 1.884 0.892 The extinction values of the ELISA are read at 495 nm

Table 2 shows that 25 mice immunized with isomes produced significantly higher antibody titers than HBs vaccines containing intact, 22 nm particles or micelles.

Test 5

In a mouse vaccination experiment using a pathogenic infection with a pathogenic rabies strain (CVS), isomers prepared according to Example 4 containing envelope proteins of rabies viruses have been studied.

25 80600

Mice were vaccinated once with the dose shown in Table 3. Mice were challenged 14 days later with a CVS strain pathogenic to mice at a dose of 40 times LDS0.

5

Table 3

Vaccination dose (pg) Number of live / dead animals 10 - 4.2 18/19 0.84 15/18 0.17 10/20 0/20 15

The table shows that isochemic vaccine confers protective immunity.

The novel antigen complexes prepared according to the invention can be stored as lyophilized preparations or as aqueous suspensions. For administration, they are suitably in a solvent such as physiological saline. 0.1 M NaCl solution (pH 7.2-7.6) is mainly used; The pH is adjusted with 0.05 M Tris-HCl. However, other buffers can also be used.

The novel protein complexes have a stronger immunogenic effect (about 10-fold or often greater) than protein micelles not prepared with a glycoside and not mixed with any adjuvant. If the corresponding protein micelles are to have the same immunogenic effects with the same dose of protein, they must be mixed with an amount of glycoside or other adjuvant so that the side effects do not become too great.

26 80 600

The protein complex prepared according to the invention does not need to be mixed with any adjuvant and thus side effects are reduced. The new complexes are still stable in contrast to antigenic proteins bound to liposomes.

Experiment 6 To 5 mg of Mycoplasma gallisepticum dissolved in 5 ml of TN buffer (pH 7.4) was added 10 mg of MEga-10. The mixture was incubated overnight at room temperature with constant stirring. Then, 5 mg of Quil A was added to the sample, and the mixture was dialyzed by changing the liquid several times against TN buffer (at room temperature for 12 hours and then at + 4 ° C). After complete dialysis, isomers containing membrane antigens are isolated by driving cell debris to the bottom after ultracentrifugation.

The EM study shows that there are isomers in the sample.

Two groups of chickens were used to demonstrate an immune response against M. gallisepticum. Each group consisted of 10 animals that were 5 weeks old at the age of 5 weeks at the start of the experiment and had an average weight of 480 g. The first group was inoculated subcutaneously with isomers containing 20 pg of protein, and the second group was left untreated as a control group. Fourteen days later, all chickens were infected with M. gallisepticum in the correct air sac of 0.1 ml at 2 x 107 cfu per inoculum. After 6 days, the chicks were sacrificed and pathologically examined for air sac damage.

In the stroke inoculated with the ischemic vaccine, the damage index decreased significantly compared to the index in the control group.

Summary: Ischemic vaccine induced protective immunity against experimental infection, and induced significant HIV titers against M. gallisepticum.

27 80600

Group Vaccine Damaged 2xt test HI titer t test PC misindex 1 isomy 9 0.001 758 0.01 5 2 control 27 - 13 -

Example 1

Parainfluenza-3 virus U 23, isolated in Umeå, 10 Sweden, is purified by centrifugation through 100,000 g of 30% by weight sucrose for one hour at 4 ° C. The precipitate is dissolved to a concentration of about 10 mg / ml in 0.05 Tris-HCl (pH 7.4) and 0.1 M NaCl (TN). 1-5 mg / ml of PI-3 virus in the same buffer 15 (TN) is dissolved in 2% by weight (final concentration) of Triton X-100 together with 3H-labeled virus at about 105 pulses / minute [ A. Luukkonen, C. Gamberg and E. Renkonen (1979), Virology 76, pp. 55-59; which is incorporated by reference]. A sample volume of approximately 200 μl was deposited on 300 μl of 15% sucrose containing 1% Triton X-100 in TN and on 12 ml of a 20-50% w / w sucrose gradient TN. in 0.2% by weight of Quil A. Centrifugation was performed at 250,000 g for 22 hours at 20 ° C. After centrifugation, fractions of 500 μl were collected from below and samples (20-50 μΐ) were measured for radioactivity. The radioactive protein fractions were pooled and dialyzed against 0.005 M Tris-HCl, 0.01 M NaCl (pH 7.4), dispensed into 10 ml flasks, and concentrated by lyophilization for 18 hours in an Edward freeze-dryer.

The sedimentation constant of this preparation is 24 S. Further purification of the complex was performed by centrifugation of the complex through a 10-40% by weight sucrose gradient.

28 80 600

Example 2

The procedure of Example 1 was repeated, but using equine influenza virus (Soiva; isolated in Solvalla, Stockholm, Sweden). The experiment was repeated without the addition of 5 glycosides (i.e. in principle according to EPD application 811 022 13.6), and the protein complexes thus obtained and the protein complex prepared with the Glycoside were used for sedimentation gradient centrifugation through a 10 to 40% by weight sugar solution at 280,000 in 10 g. For 4 hours at + 4 ° C.

The result is shown in Figure 1, which also shows the sedimentation constant of the standard thyroglobulin (indicated by an arrow, 19 S). It appears that the sedimentation constant for protein cells is 30 S and for glycoside protein enzymes it is 19 S. (Viral glycoprotein was labeled by the galactose oxidase-3 H-borohydride method.)

Example 3

The procedure of Example 1 was repeated using measles virus instead of parainfluenza-3 virus. A complex with a characteristic structure by electron microscopy is obtained, as shown in Figure 2.

Example 4

Rabies virus, prepared by RIV, in Bilthoven by the method described by Van Wezel et al .; Develop. 25 Biol. Standard (1978), 41, pages 159-158; was concentrated to 2 mg / ml in TN. 1 mg of virus was dissolved in 100 mM octyl-8-D-glycoside and incubated for 45 minutes at 20 ° C. The sample was layered on top of a 50% by weight sugar solution and centrifuged at 250,000 g for 45 minutes at + 4 ° C. The centrifugate was collected and Quil A was added up to 0.2% by weight.

The sample was sealed in a cellulose tube and dialyzed at + 20 ° C against 0.15 M ammonium acetate buffer in a volume of 1000 ml, the buffer was changed 3 times in 72 hours; during which time it was stirred continuously. The dialyzed 29 80 600 substance contains a rabies virus complex. A portion of the material was further purified by centrifugation through a 10-40% w / w sugar solution at 280,000 g for 4 hours at + 4 ° C.

5 Electron microscopy gives the structure shown in Figure 3.

Example 5

The procedure of Example 4 was repeated with measles virus. The resulting complex had the same structure as the complex prepared according to Example 3.

Example 6

Parainfluenza-3 virus (U 23) was purified by sucrose gradient centrifugation and the purified virus thus obtained was dissolved at a concentration of 10 mg / ml in 0.02 M barbiton buffer (pH 8.0) with 0.24 M Gluose (BG ), 1-5 mg / ml of PI-3 virus in BG buffer was dissolved with 2% Triton X-100 and about 105 3 H pulses / minute of labeled virus [Luukkonen et. et al., 1977) in BG buffer. A 1 ml test space was layered on a 1% agarose gel containing 0.02 M barbiturate buffer (pH 8.0) and 0.02 wt% Quil A. The agarose gel was enclosed in a tube with an area of 85 mm 2 and a height of 25 mm. The top and bottom of the tube were each connected to 0.02 M barbiturate-25 buffer (pH 8.0). A negative electrode was connected to the upper vessel. Electrophoresis was performed at 5V / cm for 4 hours. Samples were collected on the anode side of the gel and measured for radioactivity. The sample was dialyzed against 0.15 M ammonium acetate (pH 7.0) and concentrated by lyophilization.

The sedimentation constant of this preparation is about 20 S measured in the same manner as in Example 2.

Further purification of the complex was performed by centrifuging the complex through a 10-40% w / w sucrose gradient.

30 80600

Example 7

Toxoplasma gondii (obtained from Tage Waller, State Veterinary Department) was purified by filtration through a swab and centrifuged through a 30 wt% precipitate-5 rose at 100,000 g for 20 minutes at 4 ° C. The purified preparation was dissolved at a concentration of 5 mg / ml in 0.05 M Tris-HCl (pH 7.4) relative to 0.1 M NaCl (TN).

1 mg of Toxoplasma gondii was dissolved in 5% octyl β-D-glycoside and 5% saponin according to the following publication: "An investment of the antigenic structure of Toxoplasma gondii". Parasite Immulogy 1981, 3, pages 235-248; together with 3 H-labeled toxoplasma, about 105 pulses / minute (Luukkonen et al., 1977) in 15 TN buffer. A sample volume of approximately 200 μl was deposited on 300 μl of 15% sucrose containing 1% Triton X-100 in TN and on a 12 ml sucrose gradient of 20-50% sucrose. and 0.2 wt% Quil A in TN. Centrifugation was performed at 20,250,000 g at 20 ° C for about 18 hours. After centrifugation, the gradient was divided into 500 μl fractions and samples from 20 to 50 μ1 were measured for radioactivity. Two different radioactive peaks could be detected. Fractions from different peaks, each peak separately, were pooled, dialyzed and concentrated by freeze-drying as described in Example 1. The complex formed with Quil A had a lower sedimentation constant than protein micelles prepared from the same parasite.

Example 8 E. coli bacteria with plasmid pill K88 were mechanically shaken and precipitated 3 times at the isoelectric point. The material was then treated in the same manner as described in Example 1. A complex of 80600 tl having a characteristic structure was obtained, as shown in Figures 2 and 3.

Example 9

The procedure of Example 8 was repeated with a Salmo-5 nella with a pore protein. A complex having the characteristic structure shown in Figures 2 and 3 is obtained.

Example 10

Feline kidney epithelial cells infected with feline leukemia virus were treated according to the method of Example 1. A complex with the characteristic structure shown in Figures 2 and 3 was obtained.

Example 11

Renal epithelial cells transformed with 15 bovine leukemia virus were treated according to the method of Example 1. A complex with the characteristic structure shown in Figures 2 and 3 was obtained.

Example 12

Parainfluenza-3 virus U 23 was purified and the protein-20in complex was prepared according to the method described in Example 1, except that the sucrose gradient in TN with 20 to 50% by weight of sucrose contains a saponin other than Quil A.

Two commercially available saponins, 25 namely Merck "Weiss", Rhine 514 0023 and Se. Lickardt "S", Schuchart, Mtlnchen. (Saponins are pure. The manufacturer does not indicate the quality of the product. They differ from Quil A in thin-layer chromatography).

A complex with a sedimentation constant of 24 30 S and showing the same structure as the complex prepared according to Example 3 is obtained.

Example 13a

Measles virus (RIV, Bilthoven) was dissolved according to Example 3. The virus-solvent mixture was centrifuged at 1000 g for 2 hours. The resulting supernatant was dialyzed against low-salt phosphate buffer (10 mM phosphate, 50 mM NaCl, pH 7.2) and transferred to a DEAE-Sephar anion exchanger equilibrated with 10 mM phosphate buffer (pH 7.2) containing 50 mM 5 NaCl and 0.05% Triton X-100. Unabsorbed material was washed off with the same buffer. The column was then equilibrated with 10 mM phosphate buffer containing 50 mM NaCl and 0.1% Quil A. The absorbed material was eluted with the same buffer containing 300 mM NaCl.

10 The eluted material contained a complex, the characteristic structure of which is shown in Figure 2.

Example 13 5 mg of the measles virus of Example 3 was placed in a DEAE cellulose type anion exchanger. The ion exchanger 15 was stored on a 20 ml column and equilibrated with 0.01 m phosphate buffer (pH 7.2) containing 0.5% by weight of octyl β-D-glycoside. The sample material was placed in an ion exchanger, and the non-adsorbed material was washed away by rinsing with 5 column volumes of 0.01 M phosphate buffer (pH 7.2) containing 0.5% by weight of octyl-βD-glucoside, followed by adsorption. the material was eluted by adding to the column a gradient of salt from 0 to 0.5 M NaCl dissolved in 0.01 M phosphate (pH 7.2) with 0.5 wt% octyl pD glycoside. Fractions from which measles membrane protein was iden-25 were pooled and 0.1 wt% Quil A was added and dialyzed against 0.05 M ammonium acetate (pH 7.0). A complex with a characteristic structure was obtained, as shown in Figures 2 and 3.

Example 14 Hepatitis B virus particles of 22 nm obtained from the London School of Tropical Medicine and Hygiene (England) were redissolved at a concentration of 1 mg / ml in TN. 0.3 mg of protein with a particle size of 2280600 at 22 nm was dissolved in 2% v / v Triton X-100, 0.5 M NaCl, and incubated for 16 hours at + 37 ° C. The method of Example 1 is repeated.

The sedimentation constant of the obtained complex was 20 S.

The obtained complex had a structure in electron microscopic examination, which is shown in Figure 4. This structure differs from the structure shown in Figure 2 in that it consists of parts of this structure.

Example 15 3 mg of bovine diarrhea virus (diarrhea virus, BDV) in TN was dissolved by adding Triton X-100 up to 1% by volume. The mixture was stirred for 2 hours at room temperature. The virus thus dissolved was applied to a lectin column consisting of lectin Lentil coupled to Sepharose 4B (Pharmacia, Uppsala). The column was equilibrated with TN, and after the viral material was applied to the column, it was washed with 5 column volumes of TN containing 0.1% by volume of Triton X-100, followed by 10 column volumes of TN. The viral envelope proteins are released from the adsorption by applying to the column an elution buffer consisting of 0.2 M methyl α-D-mannoside and 0.5% by weight of octyl-β-D-glycoside dissolved in TN. Fractions containing viral envelope proteins are pooled and added to Quil A up to 0.1% by weight. The mixture is dialyzed against 0.05 M ammonium acetate (pH 7.0) at -t-4 ° C for 3 days, changing the volume of 1 liter of buffer 3 times.

The final product is lyophilized and electron microscopy 30 shows the (complex) structures formed by the parts of the complex in Figure 4. The sedimentation constant of this preparation is 20 S.

34 8 0 6 0 0

Example 16

Purified, killed poliovirus, prepared by RIV, in Bilthoven by the method described by Van We-zel et al. [Develop. Biol. Standard (1978), 41, pages 159 5-168], was dissolved in a suitable buffer, e.g. TN, containing a solubilizing agent, e.g. 2% SDS (sodium dodecyl sulphate), by incubation at 37 ° C for 2 hours. Virus capsid proteins were separated electrophoretically on a preparative 10% polyacrylamide gel containing 0.1% SDS. The location of the proteins in the gel was determined, after which appropriate bands were excised from the gel, and the proteins were eluted electrophoretically. VP3, with a molecular weight of about 26 kilodaltons, is a capsid protein. Triton 15 X-100 was added to the VP3-containing solution to a final concentration of 2%. The mixture was used to prepare a protein complex (isom) by the centrifugation method of Example 1.

Electron microscopic examination revealed the characteristic structure of the preparation, as shown in Figure 20 3.

Example 17

Purified, killed poliovirus, prepared in RIV Bilthoven, was dissolved in 67% v / v acetic acid containing 0.1 M MgCl 2. The viral material was then ultracentrifuged for 1 hour at 100,000 g, and the supernatant containing exclusively viral proteins was collected and dialyzed in the presence of 0.1 wt% Quil A, 0.01 M Tris. , Against 0.14 M NaCl (pH 7.4).

The obtained complex had the same structure as the complex prepared according to Example 3.

• »s5 80600

Example 18

The outer membrane proteins of Neisseria meningitidis were obtained lyophilized from the National Institute of Health (Netherlands) and dissolved in TN containing 5% by weight of octyl pD glycoside and 0.1% by weight of Quil A. and was treated in the same manner as in Example 4. A complex having a sedimentation constant of 20 S measured in the same manner as in Example 2 was obtained.

Example 19 10 Peptide containing hydrophobic amino acids.

Foot-and-mouth disease peptide 144 - 159 0 Kaufbenren.

VP1 synthesized with 0, 1, 2, 3 or 4 tyrosine, respectively, was used by combining them at one end (commercially obtained). The peptide is dissolved in a minimum of 15% v / v acetic acid, neutralized with 25% ammonia and mixed with 0.5 M ammonium acetate in distilled water (detergent to a final concentration of 2%). 0.1% glycoside is added and the mixture is dialyzed against 0.05 M ammonium acetate-20 (pH 7.0) (dialysis tube Spectra Por 6 MWCO 1,000).

Mice were vaccinated twice with 50 pg every 2 weeks.

25 Peptide / tyrosine ELISA value (antibodies) number second sample 0 0.005 1 0.217 30 2 0.311 3 0.347 4 0.346

36 8 O 6 O O

Complex formation can be demonstrated by electron microscopy (see Figure 5), which shows a spherical, electron-dense particle with a length of 20 to 40 nm and a width of 10 nm. Aberrant sizes are also visible.

Figure 5a shows an electron micrograph of a peptide coupled to three tyrosines and Figure 5b shows an electron micrograph of a peptide coupled to four tyrosines.

10 Example 20

Mouse IgG purified by known methods [M. Van der Branden, J. L. de Coen, L. Kanarek and Ruyshaert; Molecular Immunology 18, (1981), pages 621-637] or enriched with ammonium sulphate solution 15 [J. E. Conradie, M. Govender, and L. Visser; Journal of Immunological Methods, Vol. 59 (1983), pages 289-299; publications are incorporated by reference]; and dialyzed overnight against 1 liter of 0.15 M phosphate citrate (PC) buffered to pH 2 overnight in a cold room. To this is added 2% detergent (e.g., octyl-β-D-glucoside). If a detergent with a low critical micellar concentration (CMC) is used, the detergent must be changed before dialysis begins. The mixture is dialyzed against PC at pH 7. After 1 hour, quil A is adjusted to a final concentration of 0.05 5 and dialysis is continued against PC and at pH 7 for one day in a cold room. .

Complex formation is demonstrated by centrifugation through a 5-30% sucrose gradient for 3.3 hours 30 at 40,000 rpm in a Beckman SW-60 rotor. The complex is detected by a gradient by ELISA.

Claims (5)

37 80600 A method of immunogenic complex comprising antigenic proteins or peptides having hydrophobic regions, having an open spherical structure formed by circular subunits or portions of a spherical structure, and generally having a lower sedimentation constant than corresponding micelles and a higher sedimentation constant than the corresponding monomeric form of the protein or peptide 10 from amphipathic proteins or peptides or viruses, mycoplasmas, parasites or animal cells containing them, characterized in that at least one of the above components is mixed with at least one solubilizing agent to form complexes of amphipathic antigens. or between the peptides and the solubilizing agent, after which the amphipathic antigenic proteins or peptides are separated from the solubilizing agent, in the presence of the solubilizing agent, or and transferred directly to a solution of at least one triterpene saponin with hydrophobic and hydrophilic regions at a concentration of at least the critical micelle content (CMC). Process according to Claim 1, characterized in that triterpene saponins selected from the group consisting of Quillaja saponaria Molina are used. Method according to claim 1 or 2, characterized in that peptides selected from the group consisting of amphipathic peptides and non-hydrophobic peptides, wherein the non-hydrophobic peptides have been rendered hydrophobic by attaching hydrophobic groups, or amphipathic peptides containing obtaining 80 80 600 hydrophobic groups in the quench tower made accessible by mild denaturation. The method according to claim 1, characterized in that membrane proteins selected from the group consisting of Orthomyxoviridae, Paramyxoviridae, Retroviridae, Rhabdoviridae, Togaviridae, Herpesviridae, hepatitis B virus, toxoplasma membrane proteins and Picoplasma membrane proteins are used. . A method for preparing an immunogenic complex according to claim 1, characterized in that a solution containing a solubilizing agent and a sugar is used, wherein the concentration of sugar is not higher than the concentration in the upper part of the triterpene saponin-containing sugar gradient, and the concentration of solubilizing agent is kept from 0.25 to 3% by weight. 20 25 30 35 39 80 600